Biology Reference
In-Depth Information
46.
Anti-HA-K: Eu
3
þ
cryptate-conjugated mouse monoclonal antibody anti-HA,
Cisbio Bioassays, #610HAKLA.
47.
Anti-HA-d2: d2-conjugated mouse monoclonal antibody anti-HA, Cisbio
Bioassays, #610HADAA.
48.
Anti-FLAG M2-K: Eu
3
þ
cryptate-conjugated mouse monoclonal antibody
anti-FLAG, Cisbio Bioassays, #61FG2KLA.
49.
Anti-FLAG M2-d2: d2-conjugated mouse monoclonal antibody anti-FLAG,
Cisbio Bioassays, #61FG2DLA.
7.2
METHODS
7.2.1
Cell transfection
Our protocols are based on transient transfection of COS-7 cells or HEK293 cells by
electroporation as described in
Claeysen et al. (1996)
. Wash cells at 70% confluence
once in PBS, trypsinize them, and, after centrifugation, resuspend them in EP1
m
buffer with 25-500 ng of epitope-tagged receptor cDNA and 15
g empty plasmid
L of cell suspension (10
7
cells) to a 0.4 cm elec-
troporation cuvette and pulse the cells using a Gene Pulser apparatus (settings:
950
m
F and 280 V or 270 V for COS-7 or HEK293, respectively). Quickly after
the shock, dilute cells in DMEM (10
7
cells/mL) containing 10% DFBS and plate
them on 100 or 150 mm Falcon cell culture dishes or into appropriated clusters.
24 h posttransfection, process the cells to study 5-HT
4
R dimers.
m
that acts as carrier. Transfer 300
7.2.2
Detection of 5-HT
4
R dimersx by Western blot
Dimerization of 5-HT
4
receptors can be evaluated by Western blotting in denaturing
conditions. Indeed 5-HT
4
R dimers form with high affinity and resist to detergents.
Four mouse splice variants of the 5-HT
4
receptor have been described that differ in
length and composition of their C-terminus: 5-HT
4(a)
,
(b)
,
(e),
and
(f)
with 387, 388,
371, and 363 amino acids, respectively (
Claeysen et al., 1999
;
Fig. 7.1
A). We used
the difference in length of these variants to show that they can interact with each
other. We also used a truncated 5-HT
4
receptor at the residue 327:
327 that is de-
△
void of the C-terminus of the receptor (
Fig. 7.1
A).
7.2.2.1
Cell lysate and membrane preparation
Dimerization of 5-HT
4
receptors is analyzed on membrane receptor preparations.
Plate four electroporations of COS-7 cells (4
10
7
cells) in two 150 mm Falcon
dishes, for each condition. Use 500 ng of Myc-tagged 5-HT
4
R cDNA per 10
7
cells
in single transfection assays and 250 ng of each receptor per 10
7
cells in cotransfec-
tion experiments. 24 h posttransfection, wash the cells with ice-cold PBS, then add
5 mL of cold PBS per 150 mm plate, and scrape the cells on ice with a rubber po-
liceman. Transfer the content of two dishes in one 12 mL Falcon tube. After centri-
fugation for 5 min at 2400 g and at 4
C, resuspend each cell pellet in 500
m
L of Tris
